RESUMEN
BACKGROUND: Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has been implicated in multiple inflammatory and non-inflammatory diseases, including liver injury induced by acetaminophen (APAP) overdose. Multiple small molecule inhibitors of MIF have been described, including the clinically available anti-rheumatic drug T-614 (iguratimod); however, this drug's mode of inhibition has not been fully investigated. METHODS: We conducted in vitro testing including kinetic analysis and protein crystallography to elucidate the interactions between MIF and T-614. We also performed in vivo experiments testing the efficacy of T-614 in a murine model of acetaminophen toxicity. We analyzed survival in lethal APAP overdose with and without T-614 and using two different dosing schedules of T-614. We also examined MIF and MIF inhibition effects on hepatic hydrogen peroxide (H2O2) as a surrogate of oxidative stress in non-lethal APAP overdose. RESULTS: Kinetic analysis was consistent with a non-competitive type of inhibition and an inhibition constant (Ki) value of 16 µM. Crystallographic analysis revealed that T-614 binds outside of the tautomerase active site of the MIF trimer, with only the mesyl group of the molecule entering the active site pocket. T-614 improved survival in lethal APAP overdose when given prophylactically, but this protection was not observed when the drug was administered late (6 h after APAP). T-614 also decreased hepatic hydrogen peroxide concentrations during non-lethal APAP overdose in a MIF-dependent fashion. CONCLUSIONS: T-614 is an allosteric inhibitor of MIF that prevented death and decreased hepatic hydrogen peroxide concentrations when given prophylactically in a murine model of acetaminophen overdose. Further studies are needed to elucidate the mechanistic role of MIF in APAP toxicity.
Asunto(s)
Benzopiranos , Enfermedad Hepática Inducida por Sustancias y Drogas , Cromonas , Factores Inhibidores de la Migración de Macrófagos , Sulfonamidas , Ratones , Animales , Acetaminofén/efectos adversos , Peróxido de Hidrógeno/metabolismo , Modelos Animales de Enfermedad , Cinética , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Estrés Oxidativo , Hígado/metabolismoRESUMEN
The macrophage migration inhibitory factor (MIF) protein family consists of MIF and D-dopachrome tautomerase (also known as MIF-2). These homologs share 34% sequence identity while maintaining nearly indistinguishable tertiary and quaternary structure, which is likely a major contributor to their overlapping functions, including the binding and activation of the cluster of differentiation 74 (CD74) receptor to mediate inflammation. Previously, we investigated a novel allosteric site, Tyr99, that modulated N-terminal catalytic activity in MIF through a "pathway" of dynamically coupled residues. In a comparative study, we revealed an analogous allosteric pathway in MIF-2 despite its unique primary sequence. Disruptions of the MIF and MIF-2 N termini also diminished CD74 activation at the C terminus, though the receptor activation site is not fully defined in MIF-2. In this study, we use site-directed mutagenesis, NMR spectroscopy, molecular simulations, in vitro and in vivo biochemistry to explore the putative CD74 activation region of MIF-2 based on homology to MIF. We also confirm its reciprocal structural coupling to the MIF-2 allosteric site and N-terminal enzymatic site. Thus, we provide further insight into the CD74 activation site of MIF-2 and its allosteric coupling for immunoregulation.
Asunto(s)
Factores Inhibidores de la Migración de Macrófagos , Humanos , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Sitios de Unión , Inflamación , Antígenos de Diferenciación de Linfocitos B/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismoRESUMEN
Macrophage migration inhibitory factor (MIF) is an inflammatory protein with various non-overlapping functions. It is not only conserved in mammals, but it is found in parasites, fish, and plants. Human MIF is a homotrimer with an enzymatic cavity between two subunits with Pro1 as a catalytic base, activates the receptors CD74, CXCR2, and CXCR4, has functional interactions in the cytosol, and is reported to be a nuclease. There is a solvent channel down its 3-fold axis with a recently identified gating residue as an allosteric site important for regulating, to different extents, the enzymatic activity and CD74 binding and signaling. In this study we explore the consequence of converting the allosteric residue Tyr99 to cysteine (Y99C) and characterize its crystallographic structure, NMR dynamics, stability, CD74 function, and enzymatic activity. In addition to the homotrimeric variant, we develop strategies for expressing and purifying a heterotrimeric variant consisting of mixed wild type and Y99C for characterization of the allosteric site to provide more insight.
RESUMEN
The macrophage migration inhibitory factor (MIF) family of cytokines contains multiple ligand-binding sites and mediates immunomodulatory processes through an undefined mechanism(s). Previously, we reported a dynamic relay connecting the MIF catalytic site to an allosteric site at its solvent channel. Despite structural and functional similarity, the MIF homolog D-dopachrome tautomerase (also called MIF-2) has low sequence identity (35%), prompting the question of whether this dynamic regulatory network is conserved. Here, we establish the structural basis of an allosteric site in MIF-2, showing with solution NMR that dynamic communication is preserved in MIF-2 despite differences in the primary sequence. X-ray crystallography and NMR detail the structural consequences of perturbing residues in this pathway, which include conformational changes surrounding the allosteric site, despite global preservation of the MIF-2 fold. Molecular simulations reveal MIF-2 to contain a comparable hydrogen bond network to that of MIF, which was previously hypothesized to influence catalytic activity by modulating the strength of allosteric coupling. Disruption of the allosteric relay by mutagenesis also attenuates MIF-2 enzymatic activity in vitro and the activation of the cluster of differentiation 74 receptor in vivo, highlighting a conserved point of control for nonoverlapping functions in the MIF superfamily.
Asunto(s)
Oxidorreductasas Intramoleculares/genética , Oxidorreductasas Intramoleculares/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Sitio Alostérico/fisiología , Secuencia de Aminoácidos/genética , Antígenos de Diferenciación de Linfocitos B/inmunología , Antígenos de Diferenciación de Linfocitos B/metabolismo , Sitios de Unión/genética , Dominio Catalítico/genética , Cristalografía por Rayos X , Citocinas/inmunología , Citocinas/metabolismo , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Oxidorreductasas Intramoleculares/fisiología , Factores Inhibidores de la Migración de Macrófagos/fisiología , Unión Proteica/genética , Relación Estructura-ActividadRESUMEN
Pre-mRNA splicing is critical for achieving required amounts of a transcript at a given time and for regulating production of encoded protein. A given pre-mRNA may be spliced in many ways, or not at all, giving rise to multiple gene products. Numerous splicing factors are recruited to pre-mRNA splice sites to ensure proper splicing. One such factor, the 60 kDa poly(U)-binding splicing factor (PUF60), is recruited to sites that are not always spliced, but rather function as alternative splice sites. In this study, we characterized the interaction of PUF60 with a splice site from the adenovirus major late promoter (the AdML 3' splice site, AdML3'). We found that the PUF60-AdML3' dissociation constants are in the micromolar range, with the binding affinity predominantly provided by PUF60's two central RNA recognition motifs (RRMs). A 1.95 Å crystal structure of the two PUF60 RRMs in complex with AdML3' revealed a dimeric organization placing two stretches of nucleic acid tracts in opposing directionalities, which can cause looping of nucleic acid and explain how PUF60 affects pre-mRNA geometry to effect splicing. Solution characterization of this complex by light-scattering and UV/Vis spectroscopy suggested a potential 2:1 (PUF602:AdML3') stoichiometry, consistent with the crystal structure. This work defines the sequence specificity of the alternative splicing factor PUF60 at the pre-mRNA 3' splice site. Our observations suggest that control of pre-mRNA directionality is important in the early stage of spliceosome assembly, and advance our understanding of the molecular mechanism by which alternative and constitutive splicing factors differentiate among 3' splice sites.
Asunto(s)
Adenoviridae/química , Intrones , Regiones Promotoras Genéticas , Sitios de Empalme de ARN , Factores de Empalme de ARN/química , ARN Viral/química , Proteínas Represoras/química , Adenoviridae/metabolismo , Cristalografía por Rayos X , Humanos , Factores de Empalme de ARN/metabolismo , ARN Viral/metabolismo , Proteínas Represoras/metabolismoRESUMEN
CXCL13 is the cognate chemokine agonist of CXCR5, a class A G-protein-coupled receptor (GPCR) that is essential for proper humoral immune responses. Using a `methionine scanning' mutagenesis method on the N-terminus of CXCL13, which is the chemokine signaling region, it was shown that minor length alterations and side-chain substitutions still result in CXCR5 activation. This observation indicates that the orthosteric pocket of CXCR5 can tolerate these changes without severely affecting the activity. The introduction of bulk on the ligand was well tolerated by the receptor, whereas a loss of contacts was less tolerated. Furthermore, two crystal structures of CXCL13 mutants were solved, both of which represent the first uncomplexed structures of the human protein. These structures were stabilized by unique interactions formed by the N-termini of the ligands, indicating that CXCL13 exhibits substantial N-terminal flexibility while the chemokine core domain remains largely unchanged. Additionally, it was observed that CXCL13 harbors a large degree of flexibility in the C-terminal extension of the ligand. Comparisons with other published structures of human and murine CXCL13 validate the relative rigidity of the core domain as well as the N- and C-terminal mobilities. Collectively, these mutants and their structures provide the field with additional insights into how CXCL13 interacts with CXCR5.
Asunto(s)
Quimiocina CXCL13 , Receptores CXCR5 , Quimiocina CXCL13/química , Quimiocina CXCL13/metabolismo , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , Receptores CXCR5/metabolismoRESUMEN
In proteins with multiple functions, such as macrophage migration inhibitory factor (MIF), the study of its intramolecular dynamic network can offer a unique opportunity to understand how a single protein is able to carry out several nonoverlapping functions. A dynamic mechanism that controls the MIF-induced activation of CD74 was recently discovered. In this study, the regulation of tautomerase activity was explored. The catalytic base Pro1 is found to form dynamic communications with the same allosteric node that regulates CD74 activation. Signal transmission between the allosteric and catalytic sites take place through intramolecular aromatic interactions and a hydrogen bond network that involves residues and water molecules of the MIF solvent channel. Once thought to be a consequence of trimerization, a regulatory function for the solvent channel is now defined. These results provide mechanistic insights into the regulation of catalytic activity and the role of solvent channel water molecules in MIF catalysis.
Asunto(s)
Oxidorreductasas Intramoleculares/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Sitio Alostérico , Biocatálisis , Células Cultivadas , Humanos , Oxidorreductasas Intramoleculares/química , Factores Inhibidores de la Migración de Macrófagos/química , Simulación de Dinámica MolecularRESUMEN
ALDH16 is a novel family of the aldehyde dehydrogenase (ALDH) superfamily with unique structural characteristics that distinguish it from the other ALDH superfamily members. In addition to structural characteristics, there is an evolutionary-related grouping within the ALDH 16 genes. The ALDH16 isozymes in frog, lower animals, and bacteria possess a critical Cys residue in their active site, which is absent from ALDH16 in mammals and fish. Genomic analysis and plasma metabolomic studies have associated ALDH16A1 with the pathogenesis of gout in humans, although its actual involvement in this disease is poorly understood. Insight into the structure of ALDH16A1 is an important step in deciphering its function in gout. Herein, we report our efforts towards the structural characterization of Xenopus tropicalis ALDH16B1 (the homolog of human ALDH16A1) that was predicted to be catalytically-active. Recombinant ALDH16B1 was expressed in Sf9 cells and purified using affinity and size exclusion chromatography. Crystallization of ALDH16B1 was achieved by vapor diffusion. A data set was collected at 2.5â¯Å and preliminary crystallographic analysis showed that the frog ALDH16B1 crystals belong to the P 212â¯121 space group with unit cell parameters aâ¯=â¯80.48â¯Å, bâ¯=â¯89.73â¯Å, câ¯=â¯190.92â¯Å, αâ¯=â¯ßâ¯=â¯Î³â¯=â¯90.00°. Structure determination is currently in progress.
Asunto(s)
Proteínas de Xenopus , Xenopus , Aldehído Deshidrogenasa/química , Aldehído Deshidrogenasa/metabolismo , Animales , Biocatálisis , Cromatografía en Gel , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Conformación Proteica , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Proteínas de Xenopus/aislamiento & purificaciónRESUMEN
The G protein-coupled receptor (GPCR) CXCR4 is a co-receptor for HIV and is involved in cancers and autoimmune diseases. We characterized five purine or quinazoline core polyamine pharmacophores used for targeting CXCR4 dysregulation in diseases. All were neutral antagonists for wild-type CXCR4 and two were biased antagonists with effects on ß-arrestin-2 only at high concentrations. These compounds displayed various activities for a constitutively active mutant (CAM). We use the IT1t-CXCR4 crystal structure and molecular dynamics (MD) simulations to develop two hypotheses for the activation of the N1193.35A CAM. The N1193.35A mutation facilitates increased coupling of TM helices III and VI. IT1t deactivates the CAM by disrupting the coupling between TM helices III and VI, mediated primarily by residue F872.53. Mutants of F872.53 in N1193.35A CXCR4 precluded constitutive signaling and prevented inverse agonism. This work characterizes CXCR4 ligands and provides a mechanism for N1193.35A constitutive activation.
Asunto(s)
Simulación de Dinámica Molecular , Receptores CXCR4/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/metabolismo , Bencilaminas , Quimiocina CXCL12/farmacología , Ciclamas , Células HEK293 , Infecciones por VIH/metabolismo , Infecciones por VIH/patología , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Compuestos Heterocíclicos/farmacología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Mutagénesis Sitio-Dirigida , Conformación Proteica en Hélice alfa , Estructura Terciaria de Proteína , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Arrestina beta 2/metabolismoRESUMEN
We report the first reversible and selective small molecule inhibitor of pro-inflammatory protein macrophage migration inhibitory factor-2 (also known as MIF-2 or d-DT). 4-(3-Carboxyphenyl)-2,5-pyridinedicarboxylic acid (4-CPPC) shows competitive binding with a 13-fold selectivity for human MIF-2 versus human MIF-1. The crystal structure of MIF-2 complexed with 4-CPPC reveals an induced fit mechanism that is not observed in the numerous MIF-1/inhibitor complexes. Crystallographic analysis demonstrates the structural source of 4-CPPC binding and selectivity for MIF-2. 4-CPPC can be employed to reveal previously unrecognized functions of MIF-1 in biological systems in which both MIF-1 and MIF-2 are expressed, to improve our knowledge of the MIF family of proteins, and to provide new mechanistic insights that can be utilized for the development of potent and selective pharmacological modulators of MIF-2.
Asunto(s)
Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Oxidorreductasas Intramoleculares/metabolismo , Factores Inhibidores de la Migración de Macrófagos/antagonistas & inhibidores , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Piridinas/química , Piridinas/farmacología , Cristalografía por Rayos X , Humanos , Oxidorreductasas Intramoleculares/química , Factores Inhibidores de la Migración de Macrófagos/química , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica/efectos de los fármacosRESUMEN
Macrophage migration inhibitory factor (MIF) activates CD74, which leads to severe disorders including inflammation, autoimmune diseases and cancer under pathological conditions. Molecular dynamics (MD) simulations up to one microsecond revealed dynamical correlation between a residue located at the opening of one end of the MIF solvent channel, previously thought to be a consequence of homotrimerization, and residues in a distal region responsible for CD74 activation. Experiments verified the allosteric regulatory site and identified a pathway to this site via the MIF ß-strands. The reported findings provide fundamental insights on a dynamic mechanism that controls the MIF-induced activation of CD74.
Asunto(s)
Antígenos de Diferenciación de Linfocitos B/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Oxidorreductasas Intramoleculares/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Sitio Alostérico , Antígenos de Diferenciación de Linfocitos B/química , Antígenos de Histocompatibilidad Clase II/química , Humanos , Inflamación/metabolismo , Oxidorreductasas Intramoleculares/química , Factores Inhibidores de la Migración de Macrófagos/química , Simulación de Dinámica Molecular , Conformación Proteica en Lámina betaRESUMEN
CD74 is a cell-surface receptor for the cytokine macrophage migration inhibitory factor. Macrophage migration inhibitory factor binding to CD74 induces its intramembrane cleavage and the release of its cytosolic intracellular domain (CD74-ICD), which regulates cell survival. In the present study, we characterized the transcriptional activity of CD74-ICD in chronic lymphocytic B cells. We show that following CD74 activation, CD74-ICD interacts with the transcription factors RUNX (Runt related transcription factor) and NF-κB and binds to proximal and distal regulatory sites enriched for genes involved in apoptosis, immune response, and cell migration. This process leads to regulation of expression of these genes. Our results suggest that identifying targets of CD74 will help in understanding of essential pathways regulating B-cell survival in health and disease.
RESUMEN
PC2 (polycystin-2) forms a Ca(2+)-permeable channel in the cell membrane and its function is regulated by cytosolic Ca(2+) levels. Mutations in the C-terminal tail of human PC2 (HPC2 Cterm) lead to autosomal dominant polycystic kidney disease. The HPC2 Cterm protein contains a Ca(2+)-binding site responsible for channel gating and function. To provide the foundation for understanding how Ca(2+) regulates the channel through the HPC2 Cterm, we characterized Ca(2+) binding and its conformational and dynamic responses within the HPC2 Cterm. By examining hydrogen-deuterium (H-D) exchange profiles, we show that part of the coiled-coil domain in the HPC2 Cterm forms a stable helix bundle regardless of the presence of Ca(2+). The HPC2 L1EF construct contains the Ca(2+)-binding EF-hand and the N-terminal linker 1 region without the downstream coiled coil. We show that the linker stabilizes the Ca(2+)-bound conformation of the EF-hand, thus enhancing its Ca(2+)-binding affinity to the same level as the HPC2 Cterm. In comparison, the coiled coil is not required for the high-affinity binding. By comparing the conformational dynamics of the HPC2 Cterm and HPC2 L1EF with saturating Ca(2+), we show that the HPC2 Cterm and HPC2 L1EF share a similar increase in structural stability upon Ca(2+) binding. Nevertheless, they have different profiles of H-D exchange under non-saturating Ca(2+) conditions, implying their different conformational exchange between the Ca(2+)-bound and -unbound states. The present study, for the first time, provides a complete map of dynamic responses to Ca(2+)-binding within the full-length HPC2 Cterm. Our results suggest mechanisms for functional regulation of the PC2 channel and PC2's roles in the pathophysiology of polycystic kidney disease.
Asunto(s)
Calcio/metabolismo , Canales Catiónicos TRPP/química , Canales Catiónicos TRPP/metabolismo , Secuencia de Aminoácidos , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Terciaria de Proteína , Canales Catiónicos TRPP/genéticaRESUMEN
For more than 15 years, the tautomerase active site of macrophage migration inhibitory factor (MIF) and its catalytic residue Pro1 have been being targeted for the development of therapeutics that block activation of its cell surface receptor, CD74. Neither the biological role of the MIF catalytic site nor the mechanistic details of CD74 activation are well understood. The inherently unstable structure of CD74 remains the biggest obstacle in structural studies with MIF for understanding the basis of CD74 activation. Using a novel approach, we elucidate the mechanistic details that control activation of CD74 by MIF surface residues and identify structural parameters of inhibitors that reduce CD74 biological activation. We also find that N-terminal mutants located deep in the catalytic site affect surface residues immediately outside the catalytic site, which are responsible for reduction of CD74 activation.
Asunto(s)
Antígenos de Diferenciación de Linfocitos B/química , Antígenos de Diferenciación de Linfocitos B/metabolismo , Antígenos de Histocompatibilidad Clase II/química , Antígenos de Histocompatibilidad Clase II/metabolismo , Factores Inhibidores de la Migración de Macrófagos/química , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Humanos , Unión Proteica , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
Polycystin-2 (PC2) belongs to the transient receptor potential (TRP) family and forms a Ca(2+)-regulated channel. The C-terminal cytoplasmic tail of human PC2 (HPC2 Cterm) is important for PC2 channel assembly and regulation. In this study, we characterized the oligomeric states and Ca(2+)-binding profiles in the C-terminal tail using biophysical approaches. Specifically, we determined that HPC2 Cterm forms a trimer in solution with and without Ca(2+) bound, although TRP channels are believed to be tetramers. We found that there is only one Ca(2+)-binding site in the HPC2 Cterm, located within its EF-hand domain. However, the Ca(2+) binding affinity of the HPC2 Cterm trimer is greatly enhanced relative to the intrinsic binding affinity of the isolated EF-hand domain. We also employed the sea urchin PC2 (SUPC2) as a model for biophysical and structural characterization. The sea urchin C-terminal construct (SUPC2 Ccore) also forms trimers in solution, independent of Ca(2+) binding. In contrast to the human PC2, the SUPC2 Ccore contains two cooperative Ca(2+)-binding sites within its EF-hand domain. Consequently, trimerization does not further improve the affinity of Ca(2+) binding in the SUPC2 Ccore relative to the isolated EF-hand domain. Using NMR, we localized the Ca(2+)-binding sites in the SUPC2 Ccore and characterized the conformational changes in its EF-hand domain due to trimer formation. Our study provides a structural basis for understanding the Ca(2+)-dependent regulation of the PC2 channel by its cytosolic C-terminal domain. The improved methodology also serves as a good strategy to characterize other Ca(2+)-binding proteins.
Asunto(s)
Calcio/metabolismo , Erizos de Mar/metabolismo , Canales Catiónicos TRPP/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Humanos , Activación del Canal Iónico , Transporte Iónico , Modelos Moleculares , Datos de Secuencia Molecular , Agregado de Proteínas , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Erizos de Mar/química , Alineación de Secuencia , Especificidad de la Especie , Canales Catiónicos TRPP/genética , Canales Catiónicos TRPP/metabolismo , TermodinámicaRESUMEN
We report the crystal structures of two inhibitors of Plasmodium falciparum macrophage migration inhibitory factor (PfMIF) with nanomolar Ki's, analyze their interactions with the active site of PfMIF, and provide explanations regarding their selectivity of PfMIF versus human MIF. These inhibitors were also found to selectively inhibit interactions between PfMIF and the human MIF receptor CD74. The results of this study provide the framework for the development of new therapeutics that target PfMIF.
Asunto(s)
Factores Inhibidores de la Migración de Macrófagos/antagonistas & inhibidores , Factores Inhibidores de la Migración de Macrófagos/química , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/química , Antígenos de Diferenciación de Linfocitos B/metabolismo , Antimaláricos/química , Antimaláricos/farmacología , Dominio Catalítico , Cristalografía por Rayos X , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Oxidorreductasas Intramoleculares/química , Oxidorreductasas Intramoleculares/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Modelos Moleculares , Estabilidad Proteica , Proteínas Protozoarias/metabolismoRESUMEN
We report a new inflammatory activity for extracellular d-dopachrome tautomerase (D-DT), the recruitment of neutrophils to the lung on D-DT intratracheal installation of C57BL/6J mice with an EC50 of 5.6 µg. We also find that D-DT and macrophage migration inhibitory factor (MIF) have additive effects in neutrophil recruitment. Although the tautomerase site of D-DT and its homologue MIF are biophysically very different, 4-iodo-6-phenylpyrimidine (4-IPP) forms a covalent bond with Pro-1 of both proteins, resulting in a 6-phenylpyrimidine (6-PP) adduct. Recruitment of neutrophils to the lung for the 6-PP adducts of D-DT and MIF are reduced by â¼ 50% relative to the apo proteins, demonstrating that an unmodified Pro-1 is important for this activity, but there is no cooperativity in inhibition of the proteins together. The differences in the binding mode of the 6-PP adduct for D-DT was determined by crystallographic studies at 1.13 Å resolution and compared to the structure of the MIF-6-PP complex. There are major differences in the location of the 6-PP adduct to the D-DT and MIF active sites that provide insight into the lack of cooperativity by 4-IPP and into tuning the properties of the covalent inhibitors of D-DT and MIF that are necessary for the development of therapeutic small molecules against neutrophil damage from lung infections such as Pseudomonas aeruginosa in cystic fibrosis and immunocompromised patients.
Asunto(s)
Dominio Catalítico/efectos de los fármacos , Oxidorreductasas Intramoleculares/metabolismo , Pulmón/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Infiltración Neutrófila/efectos de los fármacos , Neutrófilos/citología , Pirimidinas/farmacología , Animales , Movimiento Celular , Pulmón/efectos de los fármacos , Ratones Endogámicos C57BL , Modelos MolecularesRESUMEN
Macrophage migration inhibitory factor (MIF) is a pleiotropic pro-inflammatory cytokine, which possesses a contributing role in cancer progression and metastasis and, thus, is now considered a promising anticancer drug target. Many MIF-inactivating strategies have proven successful in delaying cancer growth. Here, we report on the synthesis of ISO-66, a novel, highly stable, small-molecule MIF inhibitor, an analog of ISO-1 with improved characteristics. The MIF:ISO-66 co-crystal structure demonstrated that ISO-66 ligates the tautomerase active site of MIF, which has previously been shown to play an important role in its biological functions. In vitro, ISO-66 enhanced specific and non-specific anticancer immune responses, whereas prolonged administration of ISO-66 in mice with established syngeneic melanoma or colon cancer was non-toxic and resulted in a significant decrease in tumor burden. Subsequent ex vivo analysis of mouse splenocytes revealed that the observed decrease in tumor growth rates was likely mediated by the selective in vivo expansion of antitumor-reactive effector cells induced by ISO-66. Compared to other MIF-inactivating strategies employed in vivo, the anticancer activity of ISO-66 is demonstrated to be of equal or better efficacy. Our findings suggest that targeting MIF, via highly specific and stable compounds, such as ISO-66, may be effective for cancer treatment and stimulation of anticancer immune responses.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias del Colon/tratamiento farmacológico , Isoxazoles/uso terapéutico , Melanoma/tratamiento farmacológico , Fenoles/uso terapéutico , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Dominio Catalítico/efectos de los fármacos , Neoplasias del Colon/inmunología , Neoplasias del Colon/patología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Isoxazoles/síntesis química , Isoxazoles/química , Factores Inhibidores de la Migración de Macrófagos/antagonistas & inhibidores , Factores Inhibidores de la Migración de Macrófagos/química , Melanoma/inmunología , Melanoma/patología , Ratones , Ratones Desnudos , Neoplasias Experimentales , Fenoles/síntesis química , Fenoles/química , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Bazo/efectos de los fármacos , Bazo/metabolismoRESUMEN
The mammalian chemokine family is segregated into four families - CC, CXC, CX3C, and XC-based on the arrangement of cysteines and the corresponding disulfides. Sequencing of the Danio rerio (zebrafish) genome has identified more than double the amount of human chemokines with the absence of the CX3C family and the presence of a new family, CX. The only other family with a single cysteine in the N-terminal region is the XC family. Human lymphotactin (XCL1) has two interconverting structures due to dynamic changes that occur in the protein. Similar to an experiment with XCL1 that identified the two structural forms, we probed for multiple forms of zCXL1 using heparin affinity. The results suggest only a single form of CXL1 is present. We used sulfur-SAD phasing to determine the three-dimensional structure CXL1. Zebrafish CXL1 (zCXL1) has three disulfides that appear to be important for a stable structure. One disulfide is common to all chemokines except those that belong to the XC family, another is similar to a subset of CC chemokines containing three disulfides, but the third disulfide is unique to the CX family. We analyzed the electrostatic potential of the zCXL1 structure and identified the likely heparin-binding site for glycosaminoglycans (GAGs). zCXL1 has a similar sequence identity with human CCL5 and CXCL12, but the structure is more related to CCL5. Our structural analysis supports the phylogenetic and genomic studies on the evolution of the CXL family.
Asunto(s)
Quimiocinas/química , Quimiocinas/genética , Evolución Molecular , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Disulfuros/metabolismo , Heparina/metabolismo , Humanos , Datos de Secuencia Molecular , Unión Proteica , Homología de Secuencia de Aminoácido , Electricidad Estática , Homología Estructural de Proteína , Pez CebraRESUMEN
The chemokine CXCL12 and its shared seven-transmembrane receptors CXCR4 and CXCR7 regulate diseases including cancer, atherosclerosis, autoimmunity, and HIV infection, making these molecules promising drug targets. These molecules also control key processes in normal development and physiology, suggesting the need to selectively modulate CXCR4 and/or CXCR7 functions and signaling to reduce potential complications of long-term therapy. We previously identified two peptides that functioned as allosteric agonists driving CXCR4-dependent chemotaxis, providing key structural information to design a small number of additional peptides to investigate determinants of CXCL12 interactions and signaling through CXCR4 and CXCR7. In the current study, we show that the previously identified peptides only minimally activated CXCR4 signaling through the cytosolic adapter protein ß-arrestin 2 and do not initiate signaling to ERK1/2. By comparison, peptides with diverse N-terminal amino acid sequences effectively activated CXCR7 signaling to ß-arrestin 2. One peptide, designated as GSLW based on its N-terminal amino acids, activated CXCR7 signaling and potentiated CXCL12-CXCR7 signaling without blocking the scavenger function of CXCR7 to internalize CXCL12. These results advance our understanding of CXCR7 ligand recognition and signaling, and provide structural information to target allosteric binding sites on this receptor as chemical probes and potential therapeutic agents.